Cancer immunotherapy using virus particles

ABSTRACT

A method of treating cancer in a subject in need thereof includes administering in situ to the cancer a therapeutically effective amount of a virus or virus-like particle.

RELATED APPLICATION

This application is a Continuation-in-Part of U.S. Patent ApplicationSerial No. PCT/US2015/059675, filed Nov. 9, 2015, which claims priorityto U.S. Provisional Patent Application Ser. No. 62/076,543, filed Nov.7, 2014, U.S. Provisional Patent Application Ser. No. 62/107,617, filedJan. 26, 2015, and U.S. Provisional Patent Application Ser. No.62/159,389, filed May 11, 2015, this application also claims priority toU.S. Provisional Application Ser. No. 62/464,997, filed Jul. 21, 2016,all of which are incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with Government support under NIH Training GrantNo. 5T32AI007363-22, NIH Training Grant No. T32 HL105338, and Grant No.NIH 1 U54 CA151662 awarded by the National Institutes of Health, andGrant No. CMMI 1333651 awarded by the National Science Foundation. TheGovernment has certain rights in the invention.

BACKGROUND

Regardless of tissue of origin, metastatic cancers uniformly carry poorprognoses. Conventional chemo- and radiotherapy are largely ineffectivefor late stage disease. The emerging field of tumor immunology offersnew therapeutic options. Novel therapeutics that seek to induceanti-tumor immunity, such as immune checkpoint inhibitors, chimericantigen receptor cell therapies, and tumor-associated antigen cancervaccines show promise, but the development of immunotherapy for canceris in an early stage and it is likely that, as with other cancertherapies, immunotherapies will be combined for optimal efficacy. Eachcancer type is unique but many solid tumors metastasize to the lungs. Anoption with limited exploration is direct application ofimmunostimulatory reagents into the suspected metastatic site or anidentified tumor. This approach, in situ vaccination, can modulate thelocal microenvironment and, like therapies such as T cell checkpointblocking antibodies, can relieve immunosuppression and potentiateanti-tumor immunity against antigens expressed by the tumor.

Research into nanoparticles as cancer therapies to this point haslargely focused on them as a delivery platform: the loading of particleswith tumor-associated antigen and immune agonists for the stimulation ofanti-tumor immunity, or the loading of particles with pre-existingconventional chemotherapeutic drugs for delivery to tumors as a means toreduce toxicity. Sheen et al., Wiley Interdiscip Rev NanomedNanobiotechnol., 6(5):496-505 (2014). However, the tendency ofnanoparticles to interact with and to be ingested by innate immune cellsgives them potential as immunostimulatory, immunoregulatory andimmunostimulatory agents if they modulate the characteristics of theingesting innate immune population.

Virus-like particles (VLPs) refer to the spontaneous organization ofcoat proteins into the three dimensional capsid structure of aparticular virus. Like active viruses, these particles are in the 20-500nm size range, but they are devoid of the virus nucleic acid. VLPs havealready been deployed as antigen components of antiviral vaccinesagainst infectious counterpart viruses hepatitis B (Halperin et al.,Vaccine, 30(15):2556-63 (2012)) and human papilloma virus (Moreira etal., Hum Vaccin., 7(7):768-75 (2011)). By preventing infection withviruses that cause cancer, vaccines utilizing VLPs are currentlycontributing to reductions in cancer incidence.

Recent studies have demonstrated that VLP therapeutic efficacy extendsbeyond the specific antigen array that they carry and that they maypossess inherent immunogenic properties that can stimulate immuneresponses against infectious agents that do not carry any antigenincluded in the VLP. Rynda-Apple et al., Nanomed., 9(12):1857-68(2014)). VLPs have shown the ability to induce protective immuneresponses in the respiratory tract in mouse models of infectiousdiseases of the lungs. VLP treatment protected mice from bacterialpneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA)(Rynda-Apple et al., Am J Pathol., 181(1):196-210 (2012)) and Coxiellaburnetii (Wiley et al., PLoS ONE., 4(9):e7142 (2009)). VLPs have alsobeen shown to protect mice in various influenza models. Patterson etal., ACS Nano., 7(4):3036-44 (2013); Richert et al., Eur J Immunol.,44(2):397-408 (2014). Protective immunity in these models was associatedwith recruitment, activation, and increased antigen-processingcapabilities, formation of inducible bronchus-associated lymphoid tissue(iBALTs), and stimulation of CD4⁺ T and B lymphocytes and CD8⁺ T cells.It is important to note that these studies reported robust induction ofboth innate and adaptive immunity and that the VLPs utilized were notantigenically related to the infectious agents, yet appeared to exerttheir therapeutic effect via the inherent immunomodulatory nature of theparticles. The mechanistic basis of immunomodulation of any VLP is notknown, but it is possible that some VLPs have more of that capacity thanothers.

SUMMARY

Embodiments described herein relate to methods of treating cancer in asubject in need thereof by administering in situ to cancer of thesubject a therapeutically effective amount of a virus or virus-likeparticle. The virus or virus-like particle can be nonreplicating andnoninfectious in the subject to avoid infection of the subject. In someembodiments, the in situ administration of the virus particle can beproximal to a tumor in the subject or directly to the tumor site toprovide a high local concentration of the virus particle in the tumormicroenvironment. The method represents a type of in situ vaccination,in which application of an immunostimulatory reagent directly to thetumor modifies the tumor microenvironment so that the immune system isable to respond to the tumor.

It was found that plant virus and virus-like particles and their uniquetherapeutic features, originally observed in lung infectious diseasemodels, could be utilized for a new application: treating cancer, suchas lung cancer. Primary lung cancer is the second most common cancer inthe United States, behind only breast cancer. Additionally, most othermajors cancers frequently metastasize to the lung, including breast,bladder, colon, kidney, melanoma, and prostate. Their research focusedon melanoma due to its increasing incidence in the US and the poorprognosis for metastatic disease, which currently has a 5-year survivalof below 10%. Flaherty et al., N Engl J Med., 363(9):809-19 (2010).

In some embodiments, the virus like particle (VLP) can include eCPMV,which does not carry any nucleic acids or potato virus X (PVX) VLP.These plant viruses can be nonreplicative and can be regarded as safefrom a human health and agricultural perspective. In planta productionprevents endotoxin contamination that may be a byproduct of other VLPsystems derived from E. coli. The VLPs are scalable, stable over a rangeof temperatures (4-60° C.) and solvent:buffer mixtures. In situvaccination or administration of CPMV or PVX VLPs alone or incombination with a chemotherapeutic in a model of metastatic lungmelanoma as well as dermal melanoma and other cancers (breast, colon,ovarian), was found to have striking efficacy in treating the cancer.

The in situ vaccination approach does not rely on the virus-likeparticles as a vehicle for drug or antigen delivery, but rather on theirinherent immunogenicity. This immunogenicity appears to be uniquelypotent when the particles are inhaled or when administered throughintratumoral administration into dermal tumors or as IP administrationwhen treating disseminated, metastatic ovarian cancer. For treatment oflung tumors, the particles can be intratracheally injected into micewith established lung tumors and this immunostimulatory treatmentresults in the rejection of those tumors and systemic immunity thatprevents growth of distal tumors. The virus-like particles describedherein (e.g., CPMV or PVX) alone are able to stimulate systemicanti-tumor immunity. The virus can potentially render the lungmicroenvironment inhospitable to tumor cell seeding or continued growth.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 (A-B) provides bar graphs showing eCPMV nanoparticles areinherently immunogenic. (A) Bone marrow-derived dendritic cells (BMDCs)exposed to eCPMV produce elevated levels of pro-inflammatory cytokinesin vitro. (B) Thioglycollate-elicited primary macrophages also secretesignificantly elevated levels of the same panel of cytokines. Both celltypes were cultured for 24 hr with 20 μg eCPMV (dark gray bars) andcytokine levels were analyzed using a multiplexed luminex array.

FIGS. 2 (A-D) provides graphs showing eCPMV inhalation induces dramaticchanges in immune cell composition and cytokine/chemokine milieu inB16F10 lung tumor-bearing mice. (A) Representative FACS plots pre-gatedon live CD45⁺ cells of non-tumor-bearing mice treated with PBS (topleft) or eCPMV (top right) and B16F10 lung tumor-bearing mice treatedwith PBS (bottom left) or eCPMV (bottom right). B16F10 mice were treatedon day 7 post-B16F10 IV injection. Lungs were harvested 24 hr afterintratracheal injection of PBS or 100 ug eCPMV. Labeling indicates (i)quiescent neutrophils, (ii) alveolar macrophages, (iii) monocytic MDSCs,(iv) granulocytic MDSCs, (v) tumor-infiltrating neutrophils, and (vi)activated neutrophils. Numbers beside circled groups is % of CD45⁺cells. Arrows indicate TINs (blue) and CD11b⁺ activated neutrophils(red). Gating strategies available in supplemental data. (B) Changes ininnate cell subsets induced by eCPMV inhalation are quantified as apercentage of CD45⁺ cells (top) and total number of cells (bottom) aspresented in panel (A). (C) Representative histograms for TINs,activated neutrophils, alveolar macrophages, and monocytic MDSCsindicating uptake of Alexa488-labeled CPMV, class-II, and CD86activation markers. (D) Lungs of B16F10 lung tumor-bearing miceexhibited elevated levels of pro-inflammatory cytokines andchemoattractants when treated with eCPMV as in panel (A).

FIGS. 3 (A-D) provide graphs and images showing eCPMV inhalationprevents formation of B16F10 metastatic-like lung tumors. (A) Schematicof experimental design. (B) Photographic images of lungs from eCPMV- andPBS-treated B16F10 tumor-bearing mice on day 21 post-tumor challenge. (Cand D) B16F10 lung metastatic-like tumor foci were quantified both bynumber in (C) or by qRT-PCR assay for melanocyte-specific Tyrp1 mRNAexpression in (D).

FIGS. 4 (A-C) provide graphs showing eCPMV treatment efficacy in B16F10lung model is immune-mediated. (A) eCPMV inhalation did notsignificantly affect tumor progression when mice lack Il-12. (B)Treatment efficacy was also abrogated in the absence of Ifn-γ. (C)NOD/scid/I12R-γ^(−/−) mice lacking T, B, and NK cells also failed torespond to eCPMV inhalation therapy. (D) Depletion of neutrophils withLy6G mAb abrogates treatment efficacy.

FIGS. 5 (A-D) provide graphs and images showing eCPMV immunotherapy issuccessful in metastatic breast, flank melanoma, colon, and ovariancarcinoma models. (A) Mice challenged with 4T1 breast tumors andintratracheally injected with PBS rapidly developed (IVIS images) andsuccumbed (Kaplan-Meier) to metastatic lung tumors beginning on day 24,whereas tumor development was delayed and survival significantlyextended in mice receiving intratracheal injection of eCPMV. (B) Micebearing intradermal flank B16F10 tumors directly injected with eCPMV(arrows indicate treatment days) showed noticeably delayed tumorprogression relative to PBS-injected controls and, in half ofeCPMV-treated mice, the tumor was eliminated altogether. (C) Micebearing intradermal flank CT26 colon tumors also responded to directinjection of eCPMV (arrows indicate treatment days) with significantlydelayed growth when compared to PBS-injected controls. (D) eCPMV alsoproved successful as a therapy for ID8-Defb29/Vegf-A ovariancancer-challenged mice, significantly improving survival when injectedIP relative to PBS-injected controls.

FIGS. 6 (A-B) provide a timechart (A) and a graph (B) showing eCPMVtreatment of dermal B16F10 induces systemic anti-tumor immunity.

FIGS. 7 (A-D) are a schematic and transmission electron micrographs of(A) PVX and (B) CPMV. C+D) Tumor treatment study. Tumors were inducedwith an intradermal injection of 125,000 cells/mouse. Mice (n=3) weretreated with 100 μg of PVX or CPMV (or PBS control) once weekly,starting 8 days post-induction. Arrows indicate injection days; micewere sacrificed when tumor volumes reached 1000 mm3 (C) Tumor growthcurves shown as relative tumor volume. (D) Survival rates of treatedmice.

FIGS. 8A-E illustrate Synthesis and characterization of PVX-DOX. A)Scheme of DOX loading onto PVX. B) Agarose gel electrophoresis of PVX,PVX-DOX, and free DOX under UV light (top) and after Coomassie Bluestaining (bottom). C) TEM images of negatively stained PVX-DOX. D)UV/visible spectrum of PVX-DOX. E) Efficacy of PVX-DOX vs. DOX in B16F10cells after 24 hours exposure (MTT assay).

FIGS. 9A-C illustrate chemo-immunotherapy treatment of B16F10 tumors.Groups (n=6) were treated with PBS, PVX, DOX, PVX-DOX, or PVX+DOX. PVXwas administered at a dose of 5 mg kg⁻¹, DOX was administered at a doseof 0.065 mg kg⁻¹. Injections were repeated every other day until tumorsreached >1000 mm3. A) Tumor growth curves shown as relative tumorvolume. Statistical significance was detected comparing PVX vs. PVX+DOX.B) Survival rates of treated mice. C) Immunofluorescence imaging ofthree representative PVX-DOX tumor sections after weekly dosing ofPVX-DOX (animals received 2 doses of PVX and were collected when tumorsreached >1000 mm³. Tumors treated with PVX-DOX (rows 1-3) were sectionedand stained with DAPI (blue), F4/80 (red), and PVX (green). Scalebar=100 μm.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Inaddition, the recitations of numerical ranges by endpoints include allnumbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

As used herein, the terms “peptide,” “polypeptide” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise the sequence of aprotein or peptide. Polypeptides include any peptide or proteincomprising two or more amino acids joined to each other by peptidebonds. As used herein, the term refers to both short chains, which alsocommonly are referred to in the art as peptides, oligopeptides andoligomers, for example, and to longer chains, which generally arereferred to in the art as proteins, of which there are many types.“Polypeptides” include, for example, biologically active fragments,substantially homologous polypeptides, oligopeptides, homodimers,heterodimers, variants of polypeptides, modified polypeptides,derivatives, analogs, fusion proteins, among others. The polypeptidesinclude natural peptides, recombinant peptides, synthetic peptides, or acombination thereof. A protein may be a receptor or a non-receptor.“Apa” is aminopentanoic acid.

A “nucleic acid” refers to a polynucleotide and includespolyribonucleotides and polydeoxyribonucleotides.

“Treating”, as used herein, means ameliorating the effects of, ordelaying, halting or reversing the progress of a disease or disorder.The word encompasses reducing the severity of a symptom of a disease ordisorder and/or the frequency of a symptom of a disease or disorder.

A “subject”, as used therein, can be a human or non-human animalNon-human animals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals, as well asreptiles, birds and fish. Preferably, the subject is human.

The language “effective amount” or “therapeutically effective amount”refers to a nontoxic but sufficient amount of the composition used inthe practice of the invention that is effective to provide effectiveimaging or treatment in a subject, depending on the compound being used.That result can be reduction and/or alleviation of the signs, symptoms,or causes of a disease or disorder, or any other desired alteration of abiological system. An appropriate therapeutic amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

A “prophylactic” or “preventive” treatment is a treatment administeredto a subject who does not exhibit signs of a disease or disorder, orexhibits only early signs of the disease or disorder, for the purpose ofdecreasing the risk of developing pathology associated with the diseaseor disorder.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology of a disease or disorder for the purpose ofdiminishing or eliminating those signs.

“Pharmaceutically acceptable carrier” refers herein to a compositionsuitable for delivering an active pharmaceutical ingredient, such as thecomposition of the present invention, to a subject without excessivetoxicity or other complications while maintaining the biologicalactivity of the active pharmaceutical ingredient. Protein-stabilizingexcipients, such as mannitol, sucrose, polysorbate-80 and phosphatebuffers, are typically found in such carriers, although the carriersshould not be construed as being limited only to these compounds.

Embodiments described herein relate to methods of treating cancer in asubject in need thereof by administering in situ to the cancer atherapeutically effective amount of a virus or virus-like particle tothe subject. The virus or virus-like particle can be nonreplicating andnoninfectious in the subject to avoid infection of the subject. In someembodiments, the in situ administration of the virus particle can beproximal to a tumor in the subject or directly to the tumor site toprovide a high local concentration of the virus particle in the tumormicroenvironment.

In some embodiments, virus particles or virus-like particles (VLPs) inwhich the viral nucleic acid is not present are administered in situ tocancer of the subject. Virus-like particles lacking their nucleic acidare non-replicating and non-infectious regardless of the subject intowhich they are introduced. An example of virus-like particles is emptyCowpea Mosaic Virus particles. In other embodiments, the virus particlesinclude a nucleic acid within the virus particle. If present, thenucleic acid will typically be the nucleic acid encoding the virus.However, in some embodiments the viral nucleic acid may have beenreplaced with exogenous nucleic acid. In some embodiments, the nucleicacid is RNA, while in other embodiments the nucleic acid is DNA. A virusparticle including nucleic acid will still be nonreplicating andnoninfectious when it is introduced into a subject which it cannotinfect. For example, plant virus particles will typically benonreplicating and noninfectious when introduced into an animal subject.

In some embodiments, the virus is a plant virus. However, abacteriophage or mammalian virus can be used in some embodiments of theinvention. When a plant virus is used, in some embodiments the plantvirus is a plant picornavirus. A plant picornavirus is a virus belongingto the family Secoaviridae, which together with mammalian picornavirusesbelong to the order of the Picornavirales. Plant picornaviruses arerelatively small, non-enveloped, positive-stranded RNA viruses with anicosahedral capsid. Plant picornaviruses have a number of additionalproperties that distinguish them from other picornaviruses, and arecategorized as the subfamily secoviridae. In some embodiments, the virusparticles are selected from the Comovirinae virus subfamily Examples ofviruses from the Comovirinae subfamily include Cowpea mosaic virus,Broad bean wilt virus 1, and Tobacco ringspot virus. In a furtherembodiment, the virus particles are from the Genus comovirus. Apreferred example of a comovirus is the cowpea mosaic virus particles.

In other embodiments, the plant virus or plant virus-like particle is anAlphaflexiviridae virus or virus-like particle. The genera comprisingthe Alphaflexiviridae family include Allexivirus, Botrexvirus,Lolavirus, Mandarivirus, Potexvirus, and Sclerodarnavirus. In furtherembodiments, the plant virus particle of the vaccine composition is aPotexvirus particle. Examples of Potexvirus include Allium virus X,Alstroemeria virus X, Alternanthera mosaic virus, Asparagus virus 3,Bamboo mosaic virus, Cactus virus X, Cassava common mosaic virus,Cassava virus X, Clover yellow mosaic virus, Commelina virus X,Cymbidium mosaic virus, Daphne virus X, Foxtail mosaic virus, Hostavirus X, Hydrangea ringspot virus, Lagenaria mild mosaic virus, Lettucevirus X, Lily virus X, Malva mosaic virus, Mint virus X, Narcissusmosaic virus, Nerine virus X, Opuntia virus X, Papaya mosaic virus,Pepino mosaic virus, Phaius virus X, Plantago asiatica mosaic virus,Plantago severe mottle virus, Plantain virus X, Potato aucuba mosaicvirus, Potato virus X, Schlumbergera virus X, Strawberry mild yellowedge virus, Tamus red mosaic virus, Tulip virus X, White clover mosaicvirus, and Zygocactus virus X. In some embodiments, the plant virus likeparticle is a Potato virus X virus-like particle.

The virus or virus-like particles can be obtained according to variousmethods known to those skilled in the art. In embodiments where plantvirus particles are used, the virus particles can be obtained from theextract of a plant infected by the plant virus. For example, cowpeamosaic virus can be grown in black eyed pea plants, which can beinfected within 10 days of sowing seeds. Plants can be infected by, forexample, coating the leaves with a liquid containing the virus, and thenrubbing the leaves, preferably in the presence of an abrasive powderwhich wounds the leaf surface to allow penetration of the leaf andinfection of the plant. Within a week or two after infection, leaves areharvested and viral nanoparticles are extracted. In the case of cowpeamosaic virus, 100 mg of virus can be obtained from as few as 50 plants.Procedures for obtaining plant picornavirus particles using extractionof an infected plant are known to those skilled in the art. See WellinkJ., Meth Mol Biol, 8, 205-209 (1998). Procedures are also available forobtaining virus-like particles. Saunders et al., Virology, 393(2):329-37(2009). The disclosures of both of these references are incorporatedherein by reference.

Cancer Treatment by Virus Particle Administration

This application describes a method of treating cancer in a subject inneed thereof by administering in situ a therapeutically effective amountof a virus or virus-like particle to the subject. While not intending tobe bound by theory, it appears that the virus particles have ananticancer effect as a result of eliciting an immune response to thecancer. “Cancer” or “malignancy” are used as synonymous terms and referto any of a number of diseases that are characterized by uncontrolled,abnormal proliferation of cells, the ability of affected cells to spreadlocally or through the bloodstream and lymphatic system to other partsof the body (i.e., metastasize) as well as any of a number ofcharacteristic structural and/or molecular features. A “cancer cell”refers to a cell undergoing early, intermediate or advanced stages ofmulti-step neoplastic progression. The features of early, intermediateand advanced stages of neoplastic progression have been described usingmicroscopy. Cancer cells at each of the three stages of neoplasticprogression generally have abnormal karyotypes, includingtranslocations, inversion, deletions, isochromosomes, monosomies, andextra chromosomes. Cancer cells include “hyperplastic cells,” that is,cells in the early stages of malignant progression, “dysplastic cells,”that is, cells in the intermediate stages of neoplastic progression, and“neoplastic cells,” that is, cells in the advanced stages of neoplasticprogression. Examples of cancers are sarcoma, breast, lung, brain, bone,liver, kidney, colon, and prostate cancer. In some embodiments, thevirus particles are used to treat cancer selected from the groupconsisting of but not limited to melanoma, breast cancer, colon cancer,lung cancer, and ovarian cancer. In some embodiments, the virusparticles are used to treat lung cancer. Inhalation is a preferredmethod of administering the virus or virus-like particles when treatinglung cancer. However, inhaled virus particles are able to treat cancerbeyond the lung as a result of their ability to stimulate a systemicimmune response. For example, in some embodiments, the virus particlesare used to treat metastatic cancer which has spread to one or moresites beyond the initial point where cancer has occurred. In otherembodiments, the virus or virus-like particles can be administeredproximal to tumors in other tissues.

In some embodiments, the method can further include the step of ablatingthe cancer. Ablating the cancer can be accomplished using a methodselected from the group consisting of cryoablation, thermal ablation,radiotherapy, chemotherapy, radiofrequency ablation, electroporation,alcohol ablation, high intensity focused ultrasound, photodynamictherapy, administration of monoclonal antibodies, immunotherapy, andadministration of immunotoxins.

In some embodiments, the step ablating the cancer includes administeringa therapeutically effective amount of an anticancer agent to thesubject. Examples of anticancer agents include angiogenesis inhibitorssuch as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide;DNA intercalating or cross-linking agents such as bleomycin,carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin,melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors suchas methotrexate, 3-Amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine β-D-arabinofuranoside, 5-Fluoro-5′-deoxyuridine,5-Fluorouracil, ganciclovir, hydroxyurea, and mitomycin C; DNA-RNAtranscription regulators such as actinomycin D, daunorubicin,doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors suchas S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole1-β-D-ribofuranoside, etoposine, formestane, fostriecin, hispidin,cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, andtyrophostin AG 879, Gene Regulating agents such as5-aza-2′-deoxycitidine, 5-azacytidine, cholecalciferol,4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, alltrans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol,tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine,dolostatin 15, nocodazole, paclitaxel, podophyllotoxin, rhizoxin,vinblastine, vincristine, vindesine, and vinorelbine; and various otherantitumor agents such as 17-(allylamino)-17-demethoxygeldanamycin,4-Amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine,dichloromethylene-diphosphonic acid, leuprolide,luteinizing-hormone-releasing hormone, pifithrin, rapamycin,thapsigargin, and bikunin, and derivatives (as defined for imagingagents) thereof.

In some embodiments, the step ablating the cancer includes immunotherapyof the cancer. Cancer immunotherapy is based on therapeuticinterventions that aim to utilize the immune system to combat malignantdiseases. It can be divided into unspecific approaches and specificapproaches. Unspecific cancer immunotherapy aims at activating parts ofthe immune system generally, such as treatment with specific cytokinesknown to be effective in cancer immunotherapy (e.g., IL-2, interferon's,cytokine inducers).

In contrast, specific cancer immunotherapy is based on certain antigensthat are preferentially or solely expressed on cancer cells orpredominantly expressed by other cells in the context of malignantdisease (usually in vicinity of the tumor site). Specific cancerimmunotherapy can be grouped into passive and active approaches.

In passive specific cancer immunotherapy substances with specificity forcertain structures related to cancer that are derived from components ofthe immune system are administered to the patient. The most prominentand successful approaches are treatments with humanised or mouse/humanchimeric monoclonal antibodies against defined cancer associatedstructures (such as Trastuzumab, Rituximab, Cetuximab, Bevacizumab,Alemtuzumab). The pharmacologically active substance exerts is activityas long as a sufficient concentration is present in the body of thepatient, therefore administrations have to be repeated based onpharmacokinetic and pharmacodynamic considerations.

On the other hand, active specific cancer immunotherapy aims atantigen-specific stimulation of the patient's immune system to recogniseand destroy cancer cells. Active specific cancer immunotherapytherefore, in general, is a therapeutic vaccination approach. There aremany types of cancer vaccine approaches being pursued, such asvaccination with autologous or allogeneic whole tumor cells (in mostcases genetically modified for better immune recognition), tumor celllysates, whole tumor associated antigens (produced by means of geneticengineering or by chemical synthesis), peptides derived from proteinantigens, DNA vaccines encoding for tumor associated antigens,surrogates of tumor antigens such as anti-idiotypic antibodies used asvaccine antigens, and the like. These manifold approaches are usuallyadministered together with appropriate vaccine adjuvants and otherimmunomodulators in order to elicit a quantitatively and qualitativelysufficient immune response (many novel vaccine adjuvant approaches arebeing pursued in parallel with the development of cancer vaccines).Another set of cancer vaccine approaches relies on manipulatingdendritic cells (DC) as the most important antigen presenting cell ofthe immune system. For example, loading with tumor antigens or tumorcell lysates, transfection with genes encoding for tumor antigens andin-vivo targeting are suitable immunotherapies that can be used togetherwith the virus or virus-like particles of the invention for cancertreatment.

Cargo Molecules

In some embodiments, the virus particle is loaded with or bonded to acargo molecule. A variety of different types of cargo molecules can beloaded into or bonded to the virus particles. Cargo molecules that areloaded into the virus particle must be sufficiently small to fit withinthe virus capsid (i.e., have a size of 10 nm or less for a typicalicosahedral capsid). Preferred cargo molecules for the present inventioninclude antitumor agents. Alternately, rather than being loaded into thevirus particle, the cargo molecule can be bonded to the virus particle.A cargo molecule can be coupled to a virus particle either directly orindirectly (e.g. via a linker group). In some embodiments, the cargomolecule is directly attached to a functional group capable of reactingwith the agent. For example, a nucleophilic group, such as an amino orsulfhydryl group, can be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide). Alternatively, asuitable chemical linker group can be used. A linker group can serve toincrease the chemical reactivity of a substituent on either the agent orthe virus particle, and thus increase the coupling efficiency. Apreferred group suitable as a site for attaching cargo molecules to thevirus particle is one or more lysine residues present in the viral coatprotein.

Dosage and Formulation of Virus Particles

When used in vivo, the constructs of the invention are preferablyadministered as a pharmaceutical composition, comprising a mixture, anda pharmaceutically acceptable carrier. The loaded picornavirus may bepresent in a pharmaceutical composition in an amount from 0.001 to 99.9wt %, more preferably from about 0.01 to 99 wt %, and even morepreferably from 0.1 to 95 wt %.

The virus particles, or pharmaceutical compositions comprising theseparticles, may be administered by any method designed to provide thedesired effect. Administration may occur enterally or parenterally; forexample orally, rectally, intracisternally, intravaginally,intraperitoneally or locally. Parenteral administration methods includeintravascular administration (e.g., intravenous bolus injection,intravenous infusion, intra-arterial bolus injection, intra-arterialinfusion and catheter instillation into the vasculature), peri- andintra-target tissue injection, subcutaneous injection or depositionincluding subcutaneous infusion (such as by osmotic pumps),intramuscular injection, intraperitoneal injection, intracranial andintrathecal administration for CNS tumors, and direct application to thetarget area, for example by a catheter or other placement device.

A preferred method for administering the virus particle to a subjecthaving lung cancer is by inhalation. For example, the virus particlescan be administered intratracheally to the lung of the subject. Foradministration by inhalation, the virus particles are preferablyformulated as an aerosol or powder. Various methods for pulmonarydelivery of nanoparticles are discussed by Mansour et al., Int JNanomedicine. 2009; 4:299-319, the disclosure of which is incorporatedherein by reference.

The compositions can also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Suitable doses can vary widely depending on the therapeutic or imagingagent being used. A typical pharmaceutical composition for intravenousadministration would be about 0.1 mg to about 10 g per subject per day.However, in other embodiments, doses from about 1 mg to about 1 g, orfrom about 10 mg to about 1 g can be used. Single or multipleadministrations of the compositions may be administered depending on thedosage and frequency as required and tolerated by the subject. In anyevent, the administration regime should provide a sufficient quantity ofthe composition of this invention to effectively treat the subject.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the virusparticles into association with a pharmaceutically acceptable carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive agent into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations. The methods of the invention includeadministering to a subject, preferably a mammal, and more preferably ahuman, the composition of the invention in an amount effective toproduce the desired effect.

One skilled in the art can readily determine an effective amount ofvirus particles to be administered to a given subject, by taking intoaccount factors such as the size and weight of the subject; the extentof disease penetration; the age, health and sex of the subject; theroute of administration; and whether the administration is local orsystemic. Those skilled in the art may derive appropriate dosages andschedules of administration to suit the specific circumstances and needsof the subject. For example, suitable doses of the virus particles to beadministered can be estimated from the volume of cancer cells to bekilled or volume of tumor to which the virus particles are beingadministered.

Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until aneffect has been achieved. Effective doses of the virus particles varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic.

Examples have been included to more clearly describe particularembodiments of the invention. However, there are a wide variety of otherembodiments within the scope of the present invention, which should notbe limited to the particular examples provided herein.

EXAMPLES Example 1: In Situ Vaccination with Plant-Derived Virus-LikeNanoparticle Immunotherapy Suppresses Metastatic Cancer

Current therapies are often ineffective for metastatic cancer andemerging immunotherapies, while promising, are early in development. Insitu vaccination refers to a process in which immunostimulatory reagentsapplied directly to the tumor modify the immunosuppressivemicroenvironment so that the immune system is able to effectivelyrespond against the tumors. The inventors hypothesized that treatment oflung tumor-bearing mice with a virus-like nanoparticle could modulatethe lung immune environment and prevent the development of B16F10metastatic-like lesions. This example shows that inhalation of aself-assembling virus-like particle derived from cowpea mosaic virus(CPMV) suppresses the development of tumors in the lungs of mice afterintravenous challenge. The disparity in tumor burden between CPMV- andPBS-treated mice was pronounced, and the effect was immune-mediated asit was not seen in Ifn-γ^(−/−), Il-12^(−/−), or NOD/scid/I12Rγ^(−/−)mice. Efficacy was also lost in the absence of Ly6G⁺ cells. CPMVnanoparticles were rapidly taken up by granulocytic cells in the tumormicroenvironment and resulted in their robust activation andcytokine/chemokine production. CPMV nanoparticles are stable, nontoxic,highly scalable, and modifiable with drugs and antigens. Theseproperties, combined with their inherent immunogenicity and significantefficacy against a poorly immunogenic tumor, present CPMV as anattractive novel immunotherapy against cancer metastatic to the lung.Additionally, CPMV exhibited clear treatment efficacy in various othertumor models including dermal melanoma, metastatic breast, colon, andovarian cancers. This is the first report of a virus-like nanoparticlebeing utilized as a cancer immunotherapy with proven therapeuticefficacy.

Materials and Methods

eCPMV Production and Characterization

eCPMV capsids were produced through agroinfiltration of Nicotianabenthamiana plants with a culture of Agrobacterium tumefaciens LBA4404transformed with the binary plasmid pEAQexpress-VP60-24K, which containsgenes for the coat protein precursor VP60 and its 24K viral proteinaseto cleave it into its mature form. 6 days post infiltration, the leaveswere harvested and eCPMV extracted using established procedures. Theparticle concentration was measured using UV/vis spectroscopy(ε_(280 nm)=1.28 mg⁻¹ mL cm⁻¹), and particle integrity was determined bytransmission electron microscopy and fast protein liquid chromatography.

Mice

C57BL/6J (01055) females were purchased from the National CancerInstitute or The Jackson Laboratory. Il-12p35^(−/−) (002692),Ifn-γ^(−/−) (002287), BALB/c (000651), and NOD/scid/I12Rγ^(−/−) (005557)female mice were purchased from The Jackson Laboratory. All mousestudies were performed in accordance with the Institutional Animal Careand Use Committee of Dartmouth.

Tumor Models

The B16F10 murine melanoma cell line was obtained from Dr. David Mullins(Geisel School of Medicine at Dartmouth College, Hanover, N.H.).4T1-luciferase murine mammary carcinoma cells were provided by AshutoshChilkoti (Duke University, Durham, N.C.). B16F10, 4T1-luc, and CT26 werecultured in complete media (RPMI supplemented with 10% FBS andpenicillin/streptomycin). ID8-Defb29/Vegf-A orthotopic ovarian serouscarcinoma cells were cultured in complete media supplemented withsodium-pyruvate as previously described. Lizotte et al., Oncoimmunology,3:e28926. eCollection 2014. Cells were harvested, washed inphosphate-buffered saline (PBS), and injected in the following mannerdepending on tumor model: 1.25×10⁵ live cells injected intravenously in200 μL PBS in the tail vein (B16F10 metastatic lung), 1.25×10⁵ livecells injected intradermally in 30 μL PBS in the right flank (B16F10flank), 1×10⁵ live cells injected intradermally in 30 μL PBS in theright flank (CT26 flank), 2×10⁶ live cells injected intraperitoneally in200 μL PBS (ID8-Defb29/Vegf-A peritoneal ovarian). For 4T1-luc tumorchallenge, 1×10⁵ live cells were injected in 30 μL of PBS into the leftmammary fat pad on day 0 and the tumor was surgically removed on day 16,a day by which it is well-established that the tumor has spontaneouslymetastasized to the lung. Complete removal of primary 4T1-luc tumors wasconfirmed by bioluminescent imaging. B16F10 and ID8-Defb29/Vegf-A aresyngeneic for the C57BL6J strain, whereas CT26 and 4T1-luc are syngeneicfor the BALB/c background.

eCPMV Treatment Scheduling

WT, Il-12^(−/−), Ifn-γ^(−/−), NOD/scid/IL2Rγ^(−/−), and Ly6G-depletedmice challenged intravenously with B16F10 were intubated andintratracheally injected with 100 μg of eCPMV in 50 μL PBS on days 3,10, and 17 post-tumor challenge. For lung challenge experiments, micewere euthanized on day 21 for quantification of metastatic-like lesionsand tyrosinase expression. 4T1-luc-bearing mice were intratracheallyinjected with 20 μg of eCPMV in 50 μL PBS on day 16 (same day as primarytumor removal) and again on day 23 (day 7 post-tumor removal). B16F10flank tumors were intratumorally injected with 100 μg eCPMV on day 7post-tumor challenge once tumors had reached 10 mm² and again on day 14.CT26 flank tumors were intratumorally injected with 100 μg eCPMV on day8 post-tumor challenge once they had reached 10 mm² and again on day 15.Flank tumor diameters were measured every other day and mice wereeuthanized when tumor diameters reached 200 mm². ID8-Defb29/Vegf-A micewere injected IP with 100 μg of eCPMV weekly beginning on day 7post-tumor challenge and euthanized when they reached 35 g due toascites development.

Antibodies and Flow Cytometry

Anti-mouse antibodies were specific for CD45 (30-F11), MHC-II(M5/114.15.2), CD86 (GL-1), CD11b (M1/70), F4/80 (BM8), and Ly6G (1A8)from Biolegend and CD16/CD32 (93) from eBioscience. WT and B16F10 lungtumor-bearing mice were intratracheally injected with 100 μgAlexa-488-labeled CPMV particles 24 hr prior to euthanization. Lungswere harvested and dissociated into single cell suspension using theMiltenyi mouse lung dissociation kit (cat #130-095-927). Red blood cellswere removed using lysis buffer of 150 mM NH4C1, 10 mM KHCO3, and 0.5 mMEDTA. Flow cytometry was performed on a MACSQuant analyzer (Miltenyi).Data were analyzed using FlowJo software version 8.7.

Tyrosinase mRNA Expression Analysis

Whole lungs were dissociated and total RNA was extracted using theRNeasy kit (Qiagen, 74104). cDNA was synthesized using iScript™ cDNAsynthesis kit (Bio-Rad, 170-8891). q-PCR was performed on a CFX96™Real-Time PCR Detection System (Bio-Rad) using iQ™ SYBR® Green Supermix(Bio-Rad, 170-8882) with primers at a concentration of 0.5 μM. mRNAtranscript fold-change was calculated using the ΔΔCT method with allsamples normalized to mouse Gapdh.

Cytokine Assay

For in vivo cytokine data, total lung homogenate was harvested fromB16F10 lung tumor-bearing mice 24 hr post-inhalation of 100 μg eCPMVparticles, which was day 8 post-tumor challenge. For in vitro cytokineresults, bone marrow-derived dendritic cells (BMDCs) andthioglycollate-stimulated peritoneal macrophages, both derived fromC57BL6 mice, were cultured at 1×10⁶ cells/well in 200 μL complete mediain 96-well round-bottomed plates with either 20 μg of eCPMV or PBS.Supernatant was harvested after 24 hr incubation. Cytokines werequantified using mouse 32plex Luminex assay (MPXMCYTO70KPMX32,Millipore).

Cell Depletion

Mice were injected with mAb depleting Ly6G (clone 1A8) that waspurchased from Bio-X-Cell (cat #BE0075-1) and administered IP in dosesof 500 μg one day prior to eCPMV treatment and then once weekly for theduration of survival experiments. Greater than 95% depletion of targetcell populations in the lung was confirmed by flow cytometry.

IVIS Imaging

Mice were injected IP with 150 mg/kg of firefly D-luciferin in PBS(PerkinElmer cat #122796) and allowed to rest for 10 min. Imagining wasconducted using the Xenogen VivoVision IVIS Bioluminescent andFluorescent Imager platform and analyzed with Living Image 4.3.1software (PerkinElmer).

Statistics

Unless noted otherwise, all experiments were repeated at least 2 timeswith 4-12 biological replicates and results were similar betweenrepeats. Figures denote statistical significance of p<0.05 as *, p<0.01as **, and p<0.001 as ***. A p-value <0.05 was considered to bestatistically significant. Data for bar graphs was calculated usingunpaired Student's t-test. Error bars represent standard error of themean from independent samples assayed within the representedexperiments. Flank tumor growth curves were analyzed using two-wayANOVA. Survival experiments utilized the log-rank Mantel-Cox test forsurvival analysis. Statistical analysis was done with GraphPad Prism 4software.

Results

eCPMV Nanoparticles are Inherently Immunogenic

Previously published work with VLPs as treatments for pathogenicinfections of the respiratory tract utilized a variety of systems andreport varying degrees of immunomodulatory capacity. Additionally, thesestudies often focused on the immune responses to antigens contained inthe VLP and not the inherent immunogenicity of the particles themselves,which has not been definitively shown for VLPs. Bessa et al., Eur JImmunol. 38(1):114-26 (2008). Moreover, it is not known if some VLPs aremore stimulatory than others. For these reasons, and the inventorsproposed use of eCPMV (eCPMV refers to “empty” cowpea mosaic virusparticle devoid of RNA) as a novel immunotherapy, they first sought todetermine its inherent immunogenicity. eCPMV VLPs were added to in vitrocultures of bone marrow-derived dendritic cells (BMDCs) and primarymacrophages harvested from C57BL6 mice. Twenty-four hours of culturewith eCPMV particles induced both BMDCs (FIG. 1A) and macrophages (FIG.1B) to secrete higher levels of canonical pro-inflammatory cytokinesincluding Il-β, Il-6, Il-12p40, Cc13 (MIP1-α), and Tnf-α, leading theinventors to conclude that eCPMV is inherently immunostimulatory.

eCPMV Inhalation Radically Alters the B16F10 Lung Tumor Microenvironment

The immunomodulatory effect of eCPMV inhalation on the lungmicroenvironment was determined next, both in terms of immune cellcomposition and changes in cytokine and chemokine levels. Exposure ofnon-tumor-bearing mouse lungs to eCPMV revealed significant activationof Ly6G⁺ neutrophils 24 hours after exposure as assessed by theirupregulation of the CD11b activation marker (Costantini et al., IntImmunol., 22(10):827-38 (2010)) (FIG. 2A top panels) and CD86co-stimulatory marker. Alexa488-labeling of the particle allowed forcell tracking, which enabled the inventors to confirm that it is thisCD11b₊Ly6G⁺ activated neutrophil subset, specifically, that takes up theeCPMV.

Lungs of mice bearing B16F10 melanoma tumors revealed a more compleximmune cell composition. By day 7 the emergence of large populations ofimmunosuppressive CD11b⁺Ly6G⁻F4/80^(lo)class-II⁻SSC^(lo) monocyticmyeloid-derived suppressor cells (MDSCs) andCD11b⁺Ly6G⁺F4/80⁻class-II^(mid)SSC^(hi) granulocytic MDSCs (FIG. 2Abottom panels) could be observed. Gabrilovich et al., Nat Rev Immunol.,(4):253-68 (2012). The inventors also observed the presence of a smallpopulation of CD11b⁺Ly6G⁺class-II^(mid)CD86^(hi) cells that have beendescribed in the literature as “tumor-infiltrating neutrophils” or “N1neutrophils” that are known to exert an anti-tumor effect throughcoordination of adaptive immune responses, production of high levels ofpro-inflammatory cytokines, recruitment of T and NK cells, and directcytotoxicity to tumor cells. Fridlender et al., Cancer Cell.16(3):183-94 (2009); Mantovani et al., Nat Rev Immunol. (8):519-31(2011). Inhalation of eCPMV into B16F10-bearing lungs dramaticallyaltered the immune cell composition 24 hours after administration.Significant increases in the tumor-infiltrating neutrophil (TIN) andCD11b₊Ly6G⁺ activated neutrophils populations, as well as a reduction inCD11b⁻Ly6G⁺ quiescent neutrophils (FIG. 2A bottom panels, see arrows)were also observed. TIN and activated CD11b⁺ neutrophil populationsincreased dramatically both as a percentage of CD45⁺ cells and also intotal number (FIG. 2B). Interestingly, it is these neutrophilsubpopulations that took up the vast majority of eCPMV particles,particularly TINs that took up 10-fold more eCPMV than CD11b⁺ activatedneutrophils (FIG. 2C). Monocytic MDSC, quiescent neutrophil, andalveolar macrophage populations did not take up eCPMV, and granulocyticMDSCs displayed uneven uptake. The TIN and activated neutrophilpopulations also expressed MHC class-II, and the TINs, in particular,displayed high levels of co-stimulatory marker CD86, indicatingpotential antigen presentation and T cell priming capability.Significant changes were not observed in the numbers of monocytic orgranulocytic MDSCs.

Activation of neutrophil populations by eCPMV is consistent with datacollected from a multiplexed cytokine/chemokine array performed on wholelung homogenate of B16F10 tumor-bearing lungs treated with eCPMV or PBS(FIG. 2D). Specifically, significant increases in neutrophilchemoattractants GM-CSF, Cxcl1, Cc15, and MIP-1α and significantincreases in cytokines and chemokines known to be produced by activatedneutrophils such as GM-CSF, Il-9, Cxcl1, Cxcl9, Cxcl10, Cc12, MIP-1α,and MIP-1β were seen. Interestingly, the inventors did not observesignificant increases in levels of Il-6 or Tnf-α, which are classicalpro-inflammatory cytokines which may be detrimental in the context oflung immunobiology.

eCPMV Inhalation Suppresses B16F10 Metastatic-Like Lung TumorDevelopment

The inventors next investigated whether the inherent immunogenicity ofthe eCPMV particle in the lung could induce anti-tumor immunity in theB16F10 intravenous model of aggressive metastatic lung cancer. Indeed,weekly intratracheal injection of 100 μg of eCPMV (FIG. 3A) resulted insignificantly reduced tumor burden as assessed by both metastatic-liketumor foci number (FIGS. 3 , B and C) and tyrosinase expression (FIG.3D). Tyrosinase-related protein 1 (Tyrp1) is a melanocyte-specific gene(Zhu et al., Cancer Res., 73(7):2104-16 (2013)) whose expression in thelung is restricted to B16F10 tumor cells, which allows for thequantitative measure of tumor development and serves as a control forthe varying sizes of metastatic-like foci.

Anti-Tumor Efficacy of eCPMV Inhalation is Immune-Mediated

The inventors determined whether the immune system was required fortreatment efficacy by repeating their experimental design described inFIG. 3 in the following transgenic mice: I1-12^(−/−), Ifn-γ^(−/−), andNOD/scid/IL2Rγ^(−/−). A significant difference in lung tumor burdenbetween eCPMV- and PBS-treated mice was not observed in the absence ofIl-12 (FIG. 4A), Ifn-γ (FIG. 4B), or in NSG mice lacking T, B, and NKcells (FIG. 4C).

eCPMV accumulates in and activates neutrophils in the lungs of B16F10tumor-bearing mice (FIG. 2 , A to C). Additionally, significantincreases in neutrophil-associated cytokines and chemokines weredetected in the mouse lung following eCPMV inhalation (FIG. 2D).Therefore, neutrophils were depleted using a monoclonal antibody toassess the necessity of neutrophils for treatment efficacy. Depletion ofneutrophils from the lungs of tumor-bearing mice abrogated theanti-tumor effect that we observed in WT mice (FIG. 4D). This, combinedwith the lack of efficacy observed in Il-12^(−/−), Ifn-γ^(−/−), and NSGmice leads the inventors to conclude that the anti-tumor effect of eCPMVinhalation on B16F10 development is through immunomodulation of the lungtumor microenvironment and, in particular, requires the presence of theneutrophil compartment.

eCPMV Anti-Tumor Efficacy is not Restricted to the B16F10 IntravenousLung Model

The inventors sought to ascertain whether eCPMV treatment efficacy wasrestricted to the B16F10 metastatic lung model or if theimmunomodulatory anti-tumor effect could transfer to other models. The4T1 BALB/c syngeneic breast cancer model, which is a transferrable yettruly metastatic model, was first utilized. 4T1 tumors established inthe mammary fat pad spontaneously metastasize to the lung by day 16, atwhich point the primary tumor was surgically removed and eCPMV treatmentbegun, injecting intratracheally to affect lung tumor development. The4T1 cells also expressed luciferase, allowing the inventors to trackmetastatic lung tumor development. Mice treated intratracheally witheCPMV particles had significantly delayed lung tumor onset andsignificantly extended survival (FIG. 5A). Mice from eCPMV and PBStreatment groups had comparable primary mammary fat pad tumor burden atday of surgical removal. Therefore, differences in tumor development andsurvival were due to treatment. No mice from either group experiencedrecurrence of primary mammary fat pad tumors.

To determine whether the eCPMV particle's unambiguous efficacy in B16F10and 4T1 metastatic lung models was unique to the lung immuneenvironment, its ability to treat dermal tumors was also tested. Bothintradermal B16F10 melanoma (FIG. 5B) and CT26 colon (FIG. 5C) tumorgrowth was significantly delayed following direct injection with eCPMVand, in half of the B16F10 eCPMV-treated mice, resulted in eliminationof the tumors altogether after only two treatments (FIG. 5B).

Finally, the therapeutic effect of eCPMV was investigated in a model ofdisseminated peritoneal serous ovarian carcinoma. Conejo-Garcia et al.,Nat Med., (9):950-8 (2004). ID8-Defb29/Vegf-A-challenged mice treatedweekly with eCPMV exhibited significantly improved survival relative toPBS-treated controls (FIG. 5D). In fact, mice receiving eCPMV survivedlonger in comparison than other immunotherapies attempted in this modelincluding a live, attenuated Listeria monocytogenes strain (Lizotte etal., Oncoimmunology, 3:e28926. eCollection 2014), avirulent Toxoplasmagondii (Baird et al., Cancer Res., 73(13):3842-51 (2013)), combinationagonistic CD40 and poly(I:C) (Scarlett et al., Cancer Res.,69(18):7329-37 (2009)), or Il-10 blocking antibody (Hart et al., T CellBiol., 2:29 (2011)). It is important to note that the anti-tumor effectsof eCPMV in the tested models are not attributable to direct tumor cellcytotoxicity, as exposure to high concentrations of the particle invitro had no effect on cancer cell viability or proliferation. Thelogical conclusion is that the striking anti-tumor effect induced byeCPMV nanoparticle treatment is fully immune-mediated and translatableto a variety of tumor models across diverse anatomical sites.

eCPMV nanoparticles are immunotherapeutic to a surprisingly high degreeand clearly modulate the tumor immune environment. BMDCs and macrophagesexposed to the particles robustly secreted select pro-inflammatorycytokines (FIGS. 1A and B). eCPMV particles were produced in plants,then plant contaminants were extracted using polyvinyl-polypyrrolidone,eCPMV isolated through PEG precipitation and sucrose gradientultracentrifugation, and finally the particles concentrated byultrapelleting. Purity was checked by UV/Vis absorbance, transmissionelectron microscopy (TEM), fast protein liquid chromatography (FPLC),and SDS gel electrophoresis. Wen et al., Biomacromolecules,13(12):3990-4001 (2012). No contaminants were detected during theseprocedures. Particles were then resuspended in PBS. Additionally, eCPMVis completely devoid of RNA that could stimulate TLR3/7. The inventorstherefore conclude that the high immunogenicity of eCPMV is due to thesize, shape, or inherent immune recognition of the viral coat protein.This is unlike virus-like or protein cage nanoparticles that aremanufactured in E. coli or other systems that may contain immunogeniccontaminants like endotoxin or viral nucleic acids that are challengingto remove during the purification process.

eCPMV inhalation transforms the lung tumor microenvironment and requiresthe presence of Ly6G⁺ neutrophils, a population that is minimallyassociated with response to tumor immunotherapy. Notably, eCPMVparticles appear to specifically target Ly6G⁺ neutrophils. eCPMV hasbeen shown to bind surface vimentin on cancer cells (Steinmetz et al.,Nanomed. 6(2):351-64 (2011)) and some antigen-presenting cells (Gonzalezet al., PLoS ONE. 4(11):e7981 (2009)), although it is not known if thisis the mechanism by which lung-resident neutrophils are internalizingthe particle, or if surface expression of vimentin is a feature ofmurine neutrophils. eCPMV appears to target quiescent neutrophils andconvert them to an activated CD11b⁺ phenotype, as well as inducing therecruitment of additional CD11b⁺ activated neutrophils andCD11b⁺class-II⁺CD86^(hi) tumor-infiltrating neutrophils. In fact, thesetwo populations—activated neutrophil and tumor-infiltrating or “N1”neutrophils—are the only innate immune cell populations thatsignificantly change rapidly following eCPMV lung exposure, bothdramatically increasing as a percentage of CD45⁺ cells and in total cellnumber (FIG. 2B). Neutrophils are viewed canonically as sentinels formicrobial infection that quickly engulf and kill bacteria beforeundergoing apoptosis, yet they have an emerging roll in tumorimmunology. Although still controversial, it appears that neutrophilspossess phenotypic plasticity analogous to the M1/M2 polarizationaccepted in macrophage literature. Studies have shown infiltration of animmunosuppressive, pro-angiogenic “tumor-associated neutrophil”population in B16F10 metastatic lung, human liver cancer, sarcoma, andlung adenocarcinoma models that is correlated with enhanced tumorprogression. Alternatively, depletion of tumor-residentimmunosuppressive neutrophils, conversion of them to a pro-inflammatoryphenotype, or recruitment of activated neutrophils to infiltrate thetumor microenvironment is associated with therapeutic efficacy.Activated neutrophils can directly kill tumor cells via release ofreactive oxygen intermediates (ROI), prime CD4⁺ T cells and polarizethem to a Th1 phenotype, cross-prime CD8⁺ T cells, and modulate NK cellsurvival, proliferation, cytotoxic activity and IFN-γ production.Activated neutrophils can also produce Cxcr3 ligands Cxcl9 and Cxcl10that can recruit CD4⁺ and CD8⁺ T cells that are correlated withanti-tumor immunotherapeutic efficacy in melanoma models. The datashowing increases in immunostimulatory neutrophil populations (FIG. 2B)agrees with the cytokine data (FIG. 2D), in which increases inneutrophil chemoattractants GM-CSF, Cxcl1, Cc15, and MIP-1α andcytokines and chemokines known to be produced by neutrophils wereobserved, including GM-CSF, Il-1β, Il-9, Cxcl1, Cxcl9, Cxcl10, Cc12,MIP-1α, and MIP-1β. This data in turn agrees with the in vivo tumorprogression data showing that neutrophils are required for eCPMVanti-tumor efficacy. Interestingly, although many cytokine and chemokinelevels were elevated to a statistically significant degree followingeCPMV treatment, changes were modest when compared to the dramaticdifferences in actual tumor burden (FIG. 3 , B to D). Moreover,increases in pro-inflammatory cytokines Tnf-α or Il-6 that are known tocause tissue damage when upregulated in the lung were not observed. Itappears, therefore, that eCPMV treatment of lung tumors is effectivewithout eliciting the kind of inflammatory cytokine response that couldcause acute lung injury.

eCPMV inhalation exhibited remarkable efficacy as a monotherapy (FIG. 3) that is very clearly immune-mediated (FIG. 4 ). This is novel becausethe eCPMV particle does not directly kill tumor cells or share anyantigenic overlap with B16F10 tumors, but induces an anti-tumor responsethat requires Th1-associated cytokines Il-12 (FIG. 4A) and Ifn-γ (FIG.4B), adaptive immunity (FIG. 4C), and neutrophils (FIG. 4D). Thissuggests that the inherent immunogenicity of eCPMV, when introduced intothe lung, disrupts the tolerogenic nature of the tumor microenvironment;in essence, removing the brakes on a pre-existing anti-tumor immuneresponse that is suppressed, or allowing a de novo anti-tumor responseto develop.

This work also shows that eCPMV anti-tumor efficacy in the intravenousB16F10 metastatic lung model is not an artifact of the C57BL6 mousestrain or the B16F10 model, as eCPMV therapy works equally impressivelyin flank B16F10, ovarian carcinoma, and two BALB/c models of metastaticbreast and colon cancer (FIG. 5 ). Constitutive luciferase expression in4T1 breast carcinoma cells and intradermal challenges of B16F10 and CT26allowed us to measure tumor progression quantitatively in a manner notfeasible in the B16F10 lung model. It is in these models that weobserved a potent and immediate anti-tumor effect that significantlydelayed tumor progression and, in the cases of the B16F10 and CT26intradermal tumors, induced rapid involution of established tumors andformation of necrotic centers (FIGS. 5 , B and C) that remained confinedto within the margins of the tumors and did not appear to affectsurrounding tissue. Such early responses to eCPMV—day 3post-intratumoral injection—would indicate that eCPMV particles areinducing innate immune cell-mediated anti-tumor responses. The eCPMVnanoparticle, alone, is immunogenic and highly effective as amonotherapy. However, it can also serve as a nanocarrier for tumorantigens, drugs, or immune adjuvants, opening up the exciting possibiltythat eCPMV can be modified to deliver a payload that further augmentsand improves its immunotherapeutic efficacy.

Example 2: eCPMV Treatment of Dermal B16F10 is Inducing SystemicAnti-Tumor Immunity

As shown in FIG. 6 , the inventors established dermal melanoma tumorsand injected the eCPMV particles directly into them (100 μg/injection,arrows indicate injection days). In half of the mice this results incomplete disappearance of the tumors. In the cured mice, we then waited4 weeks and re-challenged with the same tumor cells, but we injectedtumor cells on the opposite flank. Most of those mice did not developsecondary tumors. To clarify: primary tumors were directly injected withparticles, whereas mice bearing secondary tumors received no treatment.They had to rely on systemic anti-tumor immune memory alone. The eCPMVwas applied locally in the first tumors, but induced systemic immunity.“Re-challenge” mice where those that previously had B16F10 dermal tumorsthat were direct-injected and that shrank and disappeared.

Example 3

Using a plant virus-based nanotechnology, we demonstrated that viruslike particles (VLPs) from the icosahedral virus cowpea mosaic virus(CPMV, 30 nm in diameter) stimulate a potent anti-tumor immune responsewhen applied as an in situ vaccine. Efficacy was demonstrated in mousemodels of melanoma, breast cancer, ovarian cancer, and colon cancer.Data indicate that the effect is systemic and durable, resulting inimmune-memory and protecting subjects from recurrence. While theunderlying mechanism has not been elucidated in depth, initial studies,in which VLPs were inhaled into the lungs of mice bearing B16F10 lungtumors, revealed a sub-population of lung antigen presenting cells (APC)that are MHC class II+ CD11b+ Ly6G+ neutrophils that ingest VLPs andactivate following VLP exposure. Further, the increase in thisneutrophil population is accompanied by a decrease of myeloid-derivedsuppressor cells (MDSCs) that mediate immune-suppression in the tumormicroenvironment. Here we set out to investigate the use of plantviruses as in situ vaccines and their combination with chemotherapyregimes.

Recent clinical and preclinical research indicates that the combinationof chemo- and immunotherapies can be beneficial because the therapyregimes can synergize to potentiate the therapy and improve patientoutcomes. For chemo-immuno combination treatment, the use of theanthracycline doxorubicin (DOX) could be a particularly powerfulapproach, because 6 DOX itself induces immunogenic cell death thatelicits an antitumor immune response. The immune response is induced bycalreticulin exposure on the surface of dying cells, which facilitatestumor cell phagocytosis by dendritic cells resulting in tumor antigenpresentation. Furthermore, doxorubicin-killed tumor cells recruitintratumoral CD11c+ CD11b+ Ly6Chi myeloid cells, which present tumorantigens to T lymphocytes; therefore, the combination of doxorubicinwith tumor vaccines or immunotherapies can synergize and potentiate theoverall efficacy.

In this example, we set out to address the following questions:

-   -   i) whether the flexuous particles formed by the potato virus X        (PVX) would stimulate an anti-tumor response when used as in        situ vaccine?    -   ii) whether the combination of VLP-based in situ vaccine with        DOX chemotherapy would potentiate therapeutic efficacy;        specifically we asked whether the formulation as combinatorial        nanoparticle where DOX is bound and delivered by PVX (PVX-DOX)        or the co-administration of the therapeutic regimens (PVX+DOX)        would be the most effective treatment strategy?

All studies were performed using a mouse model of melanoma.

Methods PVX and CPMV Production

PVX was propagated in Nicotiana benthamiana plants and purified aspreviously reported. CPMV was propagated in Vigna unguiculata plants andpurified as previously reported.

Synthesis of PVX-DOX

PVX (2 mg mL-1 in 0.1 M potassium phosphate buffer (KP), pH 7.0) wasincubated with a 5,000 molar excess of doxorubicin (DOX) at a 10% (v/v)final concentration of DMSO for 5 days at room temperature, withagitation. PVX-DOX was purified twice over a 30% (w/v) sucrose cushionusing ultracentrifugation (212,000×g for 3 h at 4° C.) and resuspendedovernight in 0.1 M KP, pH 7.0. PVX-DOX filaments were analyzed usingUV/visible spectroscopy, transmission electron microscopy, and agarosegel electrophoresis.

UV/Visible Spectroscopy

The number of DOX per PVX filament was determined by UV/visiblespectroscopy, using the NanoDrop 2000 spectrophotometer. DOX loading wasdetermined using 20 the Beer-Lambert law and DOX (11,500 M-1 cm-1 at 495nm) and PVX (2.97 mL mg-1 cm-1 at 260 nm) extinction coefficients.

Transmission Electron Microscopy (TEM)

TEM imaging was performed after DOX loading to confirm integrity ofPVX-DOX filaments. PVX-DOX samples (0.1 mg mL⁻¹, in dH₂O) were placed oncarbon-coated copper grids and negatively stained with 0.2% (w/v) uranylacetate. Grids were imaged using a Zeiss Libra 200FE transmissionelectron microscope, operated at 200 kV.

Agarose Gel Electrophoresis

To confirm DOX attachment, PVX-DOX filaments were run in a 0.8% (w/v)agarose gel (in TBE). PVX-DOX and corresponding amounts of free DOX orPVX alone were loaded with 6× agarose loading dye. Samples were run at100 V for 30 min in TBE. Gels were visualized under UV light and afterstaining with 0.25% (w/v) Coomassie blue.

Cell Culture and Cell Viability Assay

B16F10 cells (ATCC) were cultured in Dulbecco's modified Eagle's media(DMEM, Life Technologies), supplemented with 10% (v/v) fetal bovineserum (FBS, Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin(penstrep, Life Technologies). Cells were maintained at 37° C., 5% CO₂.Confluent cells were removed with 0.05% (w/v) trypsin-EDTA (LifeTechnologies), seeded at 2×10³ cells/100 μL/well in 96-well plates, andgrown overnight at 37° C., 5% CO₂. The next day, cells were washed 2times with PBS and incubated with free DOX or PVX-DOX corresponding to0, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM DOX for 24 h, in triplicate. APVX only control corresponded to the amount of PVX in the highestPVX-DOX sample. Following incubation, cells were washed 2 times toremove unbound DOX or particles. Fresh medium (100 μL) was added andcells were returned to the 21 incubator for 48 h. Cell viability wasassessed using an MTT proliferation assay (ATCC); the procedure was asthe manufacturer suggested.

Animal Studies

All experiments were conducted in accordance with Case Western ReserveUniversity's Institutional Animal Care and Use Committee. C57BL/6J malemice (Jackson) were used. B16F10 tumors were induced intradermally intothe right flank of C57BL/6J mice (1.25×10⁵ cells/50 μL media). Animalswere monitored and tumor volume was calculated as V=0.5×a×b2, wherea=width of the tumor and b=length of the tumor. Animals were sacrificedwhen tumor volume reached >1000 mm3. Treatment schedule: Eight dayspost-tumor induction (day 0), mice were randomly assigned to thefollowing groups (n=3): PBS, PVX, or CPMV. Mice were treatedintratumorally (20 μL), every 7 days, with 5 mg kg-1 PVX or CPMV. Micewere sacrificed when tumors reached a volume >1000 mm³. Forchemo-immunotherapy combination therapy, mice were randomly assigned tothe following groups (n=6): PBS, PVX, DOX, conjugated PVX-DOX, orPVX+DOX mixtures. PVX+DOX samples were prepared less than 30 min beforeinjections and are considered not bound to each other. Mice were treatedintratumorally (20 μL), every other day, with 5 mg kg⁻¹ PVX or PVX-DOXor the corresponding dose of DOX (0.065 mg kg-1). Mice were sacrificedwhen tumors reached a volume >1000 mm³.

Immunostaining

When tumor reached volumes <100 mm³, mice were randomly assigned to thefollowing groups (n=3): PBS or PVX-DOX. Mice were treated intratumorally(20 μL), every 7 days, with 5 mg kg-1 PVX-DOX. Mice were sacrificed whentumors reached a volume >1000 mm3 tumors were collected for analysis.Tumors were frozen in optimal cutting temperature compound (Fisher).Frozen tumors were cut into 12 μm sections. Sections were fixed in 95%(v/v) ethanol for 20 minutes on ice. Following fixation, tumor sectionswere permeabilized with 22 0.2% (v/v) Triton X-100 in PBS for 2 min atroom temperature for visualization of intracellular markers. Then, tumorsections were blocked in 10% (v/v) GS/PBS for 60 min at roomtemperature. PVX and F4/80 were stained using rabbit anti-PVX antibody(1:250 in 1% (v/v) GS/PBS) and rat anti-mouse F4/80 (1:250 in 1% (v/v)GS/PBS) for 1-2 h at room temperature. Primary antibodies were detectedusing secondary antibody staining AlexaFluor488-labeled goat-anti-rabbitantibody (1:500 in 1% (v/v) GS/PBS) and AlexaFluor555-labeledgoat-anti-rat antibody (1:500 in 1% (v/v) GS/PBS) for 60 min at roomtemperature. Tumor sections were washed 3 times with PBS in between eachstep. Following the final wash, coverslips were mounted usingFluoroshield with DAPI. Slides were imaged on a Zeiss Axio Observer Z1motorized FL inverted microscope. Fluorescence intensity was analyzedusing ImageJ 1.47d (http://imagej.nih.gov/ij).

Luminex Assay

Intradermal melanomas were induced in C57BL/6J male mice (Jackson) asdescribed above. Eight days post-tumor induction (day 0), mice wererandomly assigned to the following groups (n=4): PBS, PVX, PVX-DOX, orPVX+DOX. Mice were treated intratumorally (20 μL) once and tumors whereharvested at 24 h.p.i. Tumors were weighed and homogenized in T-PER™Buffer (ThermoFisher) at 1 mL of buffer/100 mg of tissue. TPER™ Bufferwas supplemented with cOmplete™ Protease Inhibitor Cocktail tablets(Roche) at one tablet per 8 mL of Buffer. After homogenization,homogenizer was rinsed with 0.5 mL HBSS (ThermoFisher) and added to thehomogenate. Homogenate was centrifuged at 9,000×g for 10 minutes at 2-8°C. Supernatants were frozen and kept at −80° C. until analyses.Millipore Milliplex MAP mouse 32-plex was run at the CRWU BioanalyteCore.

Results and Discussion

PVX is a filamentous plant virus, measuring 515×13 nm, and is comprisedof 1270 identical coat proteins. While different in its physical naturecompared to the 30 nm-sized icosahedrons formed by CPMV, PVX and PapMVshare similar organization of the nucleoproteins arranged as flexuoussoft matter filaments. To test whether PVX would stimulate an anti-tumorresponse when used as an in situ vaccine, we used the B16F10 melanomamodel. B16F10 is a highly aggressive and poorly immunogenic tumor modelused extensively for immunotherapy studies; it also has served as amodel for the evaluation of the immunotherapeutic potential ofvirus-based therapies. Its low immunogenicity makes it an attractiveplatform to investigate new immunostimulatory therapies. B16F10isografts were induced intradermally on the right flank of C57BL/6Jmice. Eight days post-induction (tumor starting volume <100 mm³), micewere randomized (n=3) and treated weekly intratumorally with PBS or 100μg of PVX or CPMV. Tumor volumes were measured daily and mice weresacrificed when tumors reached >1000 mm³. Treatment with CPMV or PVXalone significantly slowed tumor growth rate and extended survival timecompared to PBS (FIG. 7 ), but there was no significant differencebetween CPMV and PVX treatment. These data indicate that PVX, like CPMV,can stimulate an anti-tumor response when used as an in situ vaccine.

Having established that PVX in situ vaccination slows tumor growth, wewent ahead with a chemo-immuno combination therapy approach. Wehypothesized that the combination of chemotherapy delivery, eitherco-administered (as physical mixture, PVX+DOX) or co-delivered (ascomplexed version, PVX-DOX), would enhance the anti-tumor effect. Theunderlying idea was that the chemotherapy would debulk the tumor toprovide a burst of tumor antigens in the context of immunogenic celldeath. This fosters specific immune recognition and response to thoseantigens; in turn, VLP-mediated immune-stimulation would further augmentanti-tumor immunity and induce memory to protect from outgrowth ofmetastases and recurrence of the disease.

To obtain the PVX-DOX complex (FIG. 8A), purified PVX was loaded withDOX by incubating a 5,000 molar excess of DOX with PVX for 5 days;excess DOX was removed by ultracentrifugation. Incubation criteria wereoptimized: increasing molar excess of DOX resulted in extensiveaggregation and further increasing incubation time did not increaseloading capacity (data not shown). The PVX-DOX complex was characterizedby agarose gel electrophoresis, UV/visible spectroscopy, andtransmission electron microscopy (TEM) (FIGS. 8B-D). TEM imagingconfirmed particle integrity following DOX loading. The PVX-DOXformulation stability was confirmed after 1 month of storage at 4° C.;DOX release was not apparent and the particles remained intact (data notshown). UV/visible spectroscopy was used to determine the number of DOXattached per PVX. The Beer-Lambert law, in conjunction with PVX- and DOXspecific extinction coefficients, was used to determine theconcentrations of both PVX and DOX in solution. The ratio of DOX to PVXconcentration was then used to determine DOX loading. Each PVX wasloaded with ˜850 DOX per PVX. Agarose gel electrophoresis analysisindicated that DOX was indeed associated with PVX and not free insolution, as free DOX was not detectable in the PVX-DOX sample. Theassociation of DOX with PVX may be explained based on hydrophobicinteractions and π-π stacking of the planar drug molecules and polaramino acids.

Efficacy of the PVX-DOX complex was confirmed using B16F10 melanomacells (FIG. 8E). DOX conjugated to PVX maintained cell killing ability,although with decreased efficacy resulting in an IC₅₀ value of 0.84 μMversus 0.28 μM for free DOX. Similar trends have been reported withsynthetic and virus-based nanoparticles for DOX delivery. The reducedefficacy may be explained by reduced cell uptake and requiredendolysosomal processing when DOX is delivered by nanoparticles.

To test the hypothesis that a combination chemo-immunotherapy wouldpotentiate the efficacy of PVX alone, DOX-loaded PVX (PVX-DOX) andPVX+DOX combinations were tested in the B16F10 murine melanoma model.The combination of PVX+DOX served to test whether merely the combinationof the therapies or the co-delivery (PVX-DOX) would enhance the overallefficacy. PVX+DOX was combined less than 30 min before injection toensure that the two therapies did not have time to interact. PBS, PVXalone, and free DOX were used as controls. When tumors were <100 mm3,mice were treated every other day intratumorally with PVX-DOX, PVX+DOX,or corresponding controls (n=6). Tumor volumes were measured daily andmice were sacrificed when tumors reached >1000 mm³. PVX was administeredat 5 mg kg⁻¹ (corresponding to a dose of 0.065 mg kg-1 DOX). Clinically,doxorubicin is administered at doses of 1-10 mg kg⁻¹, intravenously. If1-10% of the injected dose reaches the tumor site, the resultingintratumoral dose would equate to 0.01-1 mg kg⁻¹; thus our intratumoraldose is within a clinically relevant range of DOX.

While there was no statistical difference in tumor growth rate orsurvival time between PVX-DOX complex versus PVX or DOX alone, PVX+DOXdid significantly slow tumor growth rate versus PVX and DOX alone (FIGS.9A+B). Thus, the data indicate that the combination of DOX chemotherapyand PVX immunotherapy indeed potentiates efficacy, however theformulation as a combined nanoparticle, PVX-DOX, did not improve thetreatment. The lack of statistically significant enhancement of efficacyof the PVX-DOX complex versus immuno- or chemo-monotherapy may beexplained by the fact that the therapies synergize best when they act ontheir own. DOX targets replicating cancer cells to induce cell death,and PVX likely associated with immune cells to stimulate an anti-tumoreffect—most likely through activation of signaling cascades throughpathogen-associated molecular pattern (PAMP) receptors and other dangersignals. Indeed, we found that PVX was co-localized with F4/80+macrophages within the tumor tissue (FIG. 9C), which may cause killingof immune cells rather than cancer cells; even if the nanoparticles donot exhibit cytotoxic effect on the immune cell population, thesequestration of PVX-DOX in the immune cells would lower the anti-tumorefficacy of the complex.

To gain insight into the underlying immunology we performedcytokine/chemokine profiling using a 32-plex MILLIPLEX® Luminex® assay.Tumors were treated with PBS, PVX, DOX, PVX-DOX, or PVX+DOX andharvested 24 hours after the first injection. Profiles were obtainedusing tumor homogenates and normalized to total protein levels by thebicinchoninic acid (BCA) assay. The PVX+DOX group repeatedly showedsignificantly higher particular cytokine and chemokine levels comparedto any other group (FIG. 10 ). Specifically, interferon gamma (IFNγ) andIFNγ-stimulated or synergistic cytokines were elevated. These included,but may not be limited to: Regulated on Activation, Normal T CellExpressed and Secreted (RANTES/CCL5), Macrophage Inflammatory Protein 1a(MIP-1a/CCL3), Monocyte Chemoattractant Protein (MCP-1/CCL2), MonokineInduced by Gamma interferon (MIG/CXCL9), and IFNγ-induced protein 10(IP-10). IFNγ is a multifunctional type II interferon critical forinducing a pro-inflammatory environment and antiviral responses and isoften associated with effective tumor immunotherapy responses. Under theinfluence of IFNγ, these chemokines mediate the influx of monocytes,macrophages, and other immune cells. Interestingly, the induction ofIFNγ was not associated with the increased expression of its masterpositive regulator, IL-12, thus in this context, the increasedexpression of IFNγ is IL-12-independent (data not shown). In the tumormicroenvironment, activation of the IFNγ pathway is in accordance withother work, where viruses were applied as an in situ vaccine.Stimulation of the IFNγ pathway alleviates the immuno-suppressive tumormicroenvironment promoting an anti-tumor immune response. The molecularreceptors and signaling cascades are yet to be elucidated, but the bodyof data indicates IFNγ to be a key player for viral-based in situvaccination approaches.

Other noteworthy cytokines/chemokines that were up-regulated includeinterleukin-1β (IL-1β) and Macrophage Colony-Stimulating Factor (M-CSF).IL-1β is known to be an early pro-inflammatory cytokine activated bymany PAMPs and Danger Associated Molecular Patterns (DAMPs). IL-1βsignaling was also observed in our earlier work with CPMV, and datasuggest that initial recognition of the viral in situ vaccine by innatesurveillance cells is promoting immune activation. IL-1β and M-CSF areboth major recruiters and activators of monocytes and macrophages to thesite of challenge. M-CSF, in particular, enhances monocyte functionsincluding phagocytic activity and cytotoxicity for tumor cells, whileinducing synthesis of inflammatory cytokines such as IL-1, TNFα, andIFNγ in monocytes. PVX monotherapy appears to follow a similar trend ofincreased expression of cytokines/chemokines, with further enhancedresponse through combination with DOX when coadministered (PVX+DOX), butreduced response when directly coupled together as PVX-DOX.

In this study, we demonstrate that PVX stimulates an anti-tumor immuneresponse when used as an in situ vaccine. Data indicate that the plantvirus-based nanoparticles activate the innate immune system locally—thisinnate immune activation is thought to overcome the immunosuppressivetumor microenvironment re-starting the cancer immunity cycle leading tosystemic elimination of cancer cells through the adaptive immune system.It is likely that innate receptors such as pattern recognition receptors(PRR) play a key role recognizing the multivalent nature of the plantvirus nanoparticles; the repetitive, multidentate coat proteinassemblies are products known as pathogen-associated molecular patterns(PAMPs).

The combination of the DOX chemotherapeutic with PVX was moreefficacious than the monotherapies when co-administered as the PVX+DOXformulation, but not when physically linked in the PVX-DOX formulation.Data show that PVX+DOX induced a higher immune mediator profile withinthe tumor microenvironment, in turn resulting in increased efficacyagainst B16F10 melanoma. A key conclusion to draw from these studies isthat the combination of chemo- and immunotherapy indeed is a powerfultool—yet the formulation of the two regimes into a single,multi-functional nanoparticle may not always be the optimal approach.

It has long been recognized that nanoparticles are preferentiallyingested by phagocytic cells. FIG. 9C confirms this and shows thatPVX-DOX is concentrated in F4/80+ macrophages within the tumor. Thebasis for reduced efficacy of PVX-DOX as compared to PVX+DOX couldsimply be because when phagocytes ingest PVX-DOX, it reduces theconcentration of DOX available to react with tumor cell DNA. If as seemslikely, the cells that respond to PVX at least initially are phagocytes,then a nonexclusive alternative could be that ingestion of PVX-DOX byphagocytes leads to a different response than ingest of PVX by itself bythose cells, thus blunting the immune response stimulated by PVX.

Chemo- and immunotherapy regimes have been recognized to synergize andseveral reports have highlighted the potential of combinatorialnanoparticles to deliver chemotherapies while stimulating the immunesystem. While the nanomedicine field strives to design multifunctionalnanoparticles that integrate several functions and therapeutic regimensinto single nanoparticle—our data indicate minimal improvement inefficacy using the combinatorial PVX-DOX nanoparticles. Significanttherapeutic efficacy with prolonged survival is only achieved when thetherapeutic regimes, PVX immunotherapy and DOX chemotherapy, areco-administered separately allowing each drug to act on their own,leading to potent anti-tumor effects.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1. A method of treating cancer in a subject in need thereof, the methodcomprising administering directly to the cancer a therapeuticallyeffective amount of an in situ vaccine, the in situ vaccine comprisingat least one of cowpea mosaic virus or cowpea mosaic virus-likeparticles, wherein the cowpea mosaic virus and cowpea mosaic virus-likeparticles are not used as a vehicle for drug or antigen delivery. 2-7.(canceled)
 8. The method of claim 1, wherein the cancer is metastaticcancer.
 9. The method of claim 1, wherein the cancer is selected fromthe group consisting of melanoma, breast cancer, colon cancer, lungcancer, and ovarian cancer.
 10. The method of claim 1, wherein thecancer is lung cancer and wherein the in situ vaccine is administered byinhalation.
 11. The method of claim 1, wherein the cancer is ovariancancer and wherein the in situ vaccine is administered byintraperitoneal administration. 12-14. (canceled)
 15. The method ofclaim 1, further comprising the step of ablating the cancer by treatingthe subject with a therapeutically effective amount of radiotherapy,chemotherapy, high intensity focused ultrasound or immunotherapy. 16.The method of claim 15, wherein the treating by chemotherapy comprisesthe administration of an anticancer agent to the subject in atherapeutically effective amount.
 17. The method of claim 15, whereinthe treating by immunotherapy comprises the administration of monoclonalantibodies to the subject in a therapeutically effective amount.
 18. Themethod of claim 1, wherein the therapeutically effective amount of thein situ vaccine is an amount effective to stimulate a systemic immuneresponse in the subject.
 19. The method of claim 1, wherein the in situvaccine comprises cowpea mosaic virus that includes a nucleic acid, andwherein said nucleic acid is RNA.
 20. The method of claim 1, wherein thesubject is a human subject.
 21. A composition for delivering an activepharmaceutical ingredient directly to a cancer in a subject in needthereof, wherein said composition comprises a pharmaceuticallyacceptable carrier and therapeutically effective amounts of: the activepharmaceutical ingredient, wherein the active pharmaceutical ingredientis an in situ vaccine that comprises at least one of cowpea mosaic virusor cowpea mosaic virus-like particles, wherein the cowpea mosaic virusand cowpea mosaic virus-like particles are not used as a vehicle fordrug or antigen delivery; and an anticancer agent.
 22. The compositionof claim 21, wherein the composition is formulated for intra-tumoralinjection.
 23. The composition of claim 21, wherein the composition isformulated as an aerosol.
 24. A unit dosage form of a formulation of anin situ vaccine and an anticancer agent; wherein the in situ vaccinecomprises at least one of cowpea mosaic virus or cowpea mosaicvirus-like particles, wherein the cowpea mosaic virus and cowpea mosaicvirus-like particles are not used as a vehicle for drug or antigendelivery.
 25. The unit dosage formulation of claim 24, wherein the insitu vaccine is in association with a liquid carrier or a finely dividedsolid carrier.
 26. A placement device for direct application of apharmaceutical composition to a cancer in a subject in need thereof,wherein said pharmaceutical composition comprises a pharmaceuticallyacceptable carrier and a therapeutically effective amount of an in situvaccine that comprises at least one of cowpea mosaic virus or cowpeamosaic virus-like particles, wherein the cowpea mosaic virus and cowpeamosaic virus-like particles are not used as a vehicle for drug orantigen delivery.
 27. The placement device of claim 26, wherein thedevice is for intra-tumoral injection of said pharmaceuticalcomposition.
 28. The placement device of claim 26, wherein the device isfor administering said pharmaceutical composition to a subject havinglung cancer by inhalation.
 29. The placement device of claim 26, whereinthe device further comprises an anticancer agent for intravenousinjection or infusion.